U.S. patent number 5,246,921 [Application Number 07/926,209] was granted by the patent office on 1993-09-21 for method for treating leukemias.
This patent grant is currently assigned to The Wistar Institute of Anatomy and Biology. Invention is credited to Premkumar Reddy, Scott Shore.
United States Patent |
5,246,921 |
Reddy , et al. |
September 21, 1993 |
**Please see images for:
( Certificate of Correction ) ** |
Method for treating leukemias
Abstract
A novel ribozyme, capable of selectively cleaving the bcr-abl
mRNA of a cell containing the Philadelphia Chromosome, thereby
blocking synthesis of BCR-ABL protein is provided. Methods for
using the ribozyme for treating leukemia patients and methods of
producing the ribozymes are also provided.
Inventors: |
Reddy; Premkumar (Philadelphia,
PA), Shore; Scott (Philadelphia, PA) |
Assignee: |
The Wistar Institute of Anatomy and
Biology (Philadelphia, PA)
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Family
ID: |
27067533 |
Appl.
No.: |
07/926,209 |
Filed: |
August 5, 1992 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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544199 |
Jun 26, 1990 |
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Current U.S.
Class: |
514/44R;
435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C07K
14/82 (20130101); C12N 15/1135 (20130101); A61K
38/00 (20130101); C12N 2310/121 (20130101); C12N
2310/111 (20130101) |
Current International
Class: |
C07K
14/82 (20060101); C12N 15/11 (20060101); A61K
38/00 (20060101); A61A 031/70 (); C07H 017/00 ();
C12P 021/06 (); C12N 015/00 () |
Field of
Search: |
;536/27,23.2
;435/69.1,172.3,328.1 ;514/44 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO8905852 |
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Jun 1989 |
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WO |
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WO9104319 |
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Apr 1991 |
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WO |
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WO9104324 |
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Apr 1991 |
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WO |
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WO9118012 |
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Nov 1991 |
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WO |
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WO9118625 |
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Dec 1991 |
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WO |
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WO9118913 |
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Dec 1991 |
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WO |
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WO9200080 |
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Jan 1992 |
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WO |
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Other References
N Sarver et al, Science, 247:1222-1225 (1990). .
J. Haseloff et al, Nature, 334:585-591 (1988). .
D. E. Ruffner et al, Gene, 82:31-41 (1989). .
M. Koizumi et al. FEBS Letters, 239(2):285-288 (1988)..
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Primary Examiner: Rollins; John W.
Attorney, Agent or Firm: Howson and Howson
Parent Case Text
This application is a continuation of U.S. patent application Ser.
No. 07/544,199, filed Jun. 26, 1990, now abandoned.
Claims
What is claimed is:
1. A synthetic RNA molecule useful for the treatment of a leukemia
caused by the presence of a hybrid oncogene resulting from a
chromosomal translocation, said RNA molecule comprising a single
strand of ribonucleic acids comprising a sequence complementary to
a sequence of the hybrid gene transcript 5' to the breakpoint of
the translocation and capable of hybridizing thereto, a second
sequence complementary to a sequence of the hybrid gene transcript
3' to the breakpoint of the translocation and capable of
hybridizing thereto, and a sequence therebetween encoding a
ribozyme capable of cleaving the hybrid gene transcript at or near
the breakpoint, thereby blocking synthesis of the tumorigenic
protein, said ribozyme comprising the nucleotide sequence: 3'
A-A-G-C-A-G-G-A-G-U-G-C-C-U-G-A-G-U-A-G-U-C-5', wherein U is
Uridine, C is Cytosine, G is Guanidine, and A is Adenine.
2. The RNA molecule according to claim 1 comprising the nucleotide
sequence: 3'
U-C-G-U-C-U-C-A-A-A-G-C-A-G-G-A-G-U-G-C-C-U-G-A-G-U-A-G-U-C-G-U-U-U-U-C-G-
G-G-A-5', wherein U is Uridine, C is Cytosine, G is Guanidine, and
A is Adenine.
3. The RNA molecule according to claim 1 wherein the oncogene is
bcr-abl, and the protein is BCR-ABL.
4. A method for treating a patient having a leukemia characterized
by the presence of a hybrid oncogene resulting from a chromosomal
translocation comprising contacting cells of a patient suffering
from leukemia with an amount of the RNA molecule of claim 1
sufficient for this RNA molecule to cleave the oncogene RNA
transcript present in the cells, effectively blocking synthesis of
a tumorigenic protein.
5. The method according to claim 4 wherein the cells are contacted
with said molecule in vitro.
6. The method according to claim 4 wherein the cells are contacted
with said molecule in vitro and subsequently re-introduced into the
patient.
7. The method according to claim 4 wherein the cells are contacted
with said molecule in vitro.
8. The method according to claim 4 wherein the leukemia is CML, ALL
or contains an oncogenic gene product which is generated by the
fusion of at least two genes.
9. The method according to claim 4 wherein the cells of the patient
are contacted with said RNA molecule which is present in a
recombinant vector.
10. A method for the treatment of disease associated with the
expression of the Philadelphia Chromosome, comprising
administration of the RNA molecule of claim 1.
11. The method according to claim 4 wherein the cells of the
patient are contacted with said RNA molecule which is present in a
lipid composition.
12. A recombinant vector comprising DNA which when expressed will
yield the RNA molecule of claim 1.
13. The vector according to claim 12 capable of expressing the RNA
molecule continually inside the cell.
14. The vector of claim 12 selected from the group consisting of a
retroviral vector, an adenoviral vector, and a vaccinia vector.
15. A recombinant vector carrying the RNA molecule of claim 1 and
capable of expressing the molecule in vitro.
16. The vector according to claim 15 comprising a vector selected
from the group consisting of a mammalian vector, a bacterial
vector, a yeast vector, a fungal vector, and an insect vector.
Description
This invention refers generally to the treatment of cancers, and
more specifically to methods for treating leukemias characterized
by the presence of a chimeric protein such as those produced by a
genetic translocation.
BACKGROUND OF THE INVENTION
Philadelphia chromosome is a hybrid chromosome resulting from a
chromosomal translocation in which a small portion of the long arm
of chromosome 9 is transferred to the long arm of chromosome 22.
This chromosomal abnormality consistently associates with human
chronic myelogenous leukemia (CML). CML is a disorder of
hematopoietic cells which results in marked proliferation of
granulocytic cells and often megakaryocytes.
Recent studies indicate that more than 95% of CML patients as well
as 15-25% of ALL (Acute Lymphocytic Leukemia) patients harbor
Philadelphia Chromosome which is designated as Ph+. Molecular
studies by several groups demonstrate that during the formation of
Philadelphia Chromosome, a portion of the c-abl gene is
translocated from chromosome 9q34 to chromosome 22q11.
Significantly, this translocation disrupts two genes, c-abl of
chromosome 9 and the bcr gene of chromosome 22, resulting in the
generation of a new, fused gene comprising portions of bcr and
c-abl.
This chimeric gene, termed bcr-abl, produces a new protein, BCR-ABL
which has several unique properties and appears to be the causative
agent of the cancers with which it is associated (See FIGS. 1A
through 1E). BCR-ABL protein is present only in tumor cells and its
synthesis in these tumor cells is believed to be related to
tumorigenicity.
Presently CML and ALL patients are treated chemotherapeutically
with conventional therapeutics and radiation. Such treatment is
plagued by well-known side-effects and is often of limited effect.
No effective treatment for these leukemias is known. Thus, other
compositions and methods for treating such cancers are being
sought.
Certain naturally occurring RNA molecules, called ribozymes,
possess the property of self-catalyzed cleavage. This reaction is
shared by a number of small circular molecules which replicate in
plants, either viroid RNAs, such as the avocado sunblotch viroid
(ASBV) or satellite RNAs which are dependent on helper viruses,
such as the satellite RNAs of tobacco ringpost virus and lucerne
transient streak virus [Haseloff et al, Nature, 334:585-591
(1988)].
Comparison of several self-cleaving RNA sequences has led to the
identification of a consensus secondary structure, termed
"hammerhead", containing 11-13 conserved nucleotides at the
junction of three helices that are precisely positioned with
respect to the cleavage site. A hammerhead of less than 60
contiguous nucleotides was found to be sufficient for rapid
cleavage in the absence of any protein [D. E. Ruffner et al, Gene,
82:31-41 (1989)]. Natural catalytic centers may be formed by
contiguous regions in the RNA [P. Keese et al, in Viroids and
Viroid-Like Pathogens, J. S. Semancik, ed. (CRC Press, Boca Raton,
Fla., 1987), pp. 1-47; A. C. Forster et al, Cell. 49:211 (1987)] or
by regions separated by a large number of nucleotides [C. J.
Hutchines et al, Nucleic Acids Res., 14: 3627 (1986); L. Epstein et
al, Cell, 48: 535 (1987)]. Cleavage occurs 3' to the GUX triplet
where X can be C, U, or A [O. C. Uhlenbeck, Nature, 328: 596
(1987); C. C. Sheldon et al, Nucleic Acids Res., 17:5679 (1989)].
The essential constituents for the hammerhead can be on separate
molecules, with one strand serving as a catalyst and the other as a
substrate. Furthermore, RNA catalytic sequences require the
conserved cleavage domain (GUX) to serve as the compatible
substrates [Haseloff et al, supra].
One such hammerhead ribozyme, consisting of three stems or helices
and a catalytic center containing 11-13 conserved nucleotides
(5'-GAAAC(N).sub.n GUN(N).sub.n CUGA(N)GA-3'), has been employed to
cleave HIV I gag transcripts [N. Sarver et al, Science,
247:1222-1225 (1990)].
There remains a need in the art for effective therapeutic
compositions and methods to treat leukemia or ameliorate its effect
on a human patient.
SUMMARY OF THE INVENTION
The present invention provides therapeutic compositions and methods
for the treatment of leukemias, which are characterized by the
presence of a chimeric protein which results from a chromosomal
translocation.
In one aspect, the invention provides a composition comprising a
synthetic RNA molecule, useful for the treatment of a leukemia
characterized by the presence of a hybrid gene resulting from a
chromosomal translocation coding for a protein which confers
tumorigenicity to a human cell. The synthetic molecule comprises a
single strand of ribonucleic acids comprising a sequence
complementary to a sequence of the coding strand of the hybrid gene
5' to the breakpoint of the translocation and capable of
hybridizing thereto, a second sequence complementary to a sequence
of the coding strand of the hybrid gene 3' to the breakpoint of the
translocation and capable of hybridizing thereto, and a sequence
therebetween encoding a ribozyme capable of cleaving the hybrid
gene at or near the breakpoint. The ribozyme sequence is preferably
a hammerhead motif.
In another aspect the invention provides a composition comprising a
synthetic RNA molecule, useful for the treatment of CML
characterized by the presence of the hybrid gene bcr-abl coding for
the BCR-ABL protein which confers tumorigenicity to a human cell.
The synthetic RNA molecule comprises a single strand of ribonucleic
acids comprising a sequence complementary to a sequence of the
coding strand of the bcr-abl gene 5' to the breakpoint of the
translocation and capable of hybridizing thereto, a second sequence
complementary to a sequence of the coding strand of the hybrid gene
3' to the breakpoint of the translocation and capable of
hybridizing thereto, and a sequence therebetween encoding a
ribozyme capable of cleaving the hybrid chromosome at or near the
breakpoint. These molecules may be synthesized by conventional
means, or expressed from a recombinant expression vector.
A further aspect of the present invention provides recombinant
vectors carrying the synthetic RNA molecules described above.
Vectors of this invention are capable of expressing the RNA
molecule and delivering the RNA molecule into a cell of a leukemic
patient. These vectors are preferably recombinant retroviral
vectors which have been altered to eliminate their pathogenicity.
Other mammalian vectors may also be employed for this purpose which
are capable of delivering the RNA molecule to the cell without
otherwise damaging the cells.
Still another aspect of the present invention provides a method of
treating leukemia with the above-described molecules and
recombinant vectors. This method entails contacting cells of a
patient suffering from leukemia with an effective amount of the
synthetic RNA molecule. Once in the cell, the synthetic RNA
molecule binds repeatedly to copies of the hybrid gene and the
ribozyme cleaves the gene in a catalytic manner, rendering it
incapable of coding for the chimeric protein.
Other aspects and advantages of the present invention are disclosed
in the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A illustrates chromosomes 9 and 22. FIG. 1B pictorially
illustrates the formation of the Philadelphia chromosome from a
translocation event between chromosomes 9 and 22. FIG. 1C
schematically illustrates the c-abl gene on chromosome and the bcr
gene of chromosome 22, and the location of the translocation. FIG.
1D schematically illustrates the chimeric chromosome where 3' bcr
sequences have been replaced by c-abl sequences. FIG. 1E
pictorially illustrates the Philadelphia chromosome, consisting of
an approximately 8 Kb bcr-abl fused mRNA, which results in the
expression of an approximately 210 Kd hybrid bcr-abl protein.
FIG. 2 illustrates the structure and sequence of one such synthetic
ribozyme RNA molecule of this invention and the bcr-abl RNA
substrate or "hammerhead" molecule. As is shown in FIG. 2, this RNA
molecule is capable of forming duplexes with bcr-abl RNA.
FIG. 3 illustrates an autoradiogram demonstrating the effects of
the RNA molecule on the bcr-abl gene transcript. Lane 2 contains
the .sup.32 P labelled bcr-abl RNA transcript and MgCl.sub.2 as a
control; Lane 3 contains MgCl.sub.2 and the unlabelled ribozyme as
a control; Lane 4 contains EDTA and the labelled bcr-abl transcript
as a control; Lanes 5 and 6 each contain MgCl.sub.2, the ribozyme
molecule and the labeled bcr-abl transcript; Lane 7 contains EDTA,
the ribozyme molecule and the labeled bcr-abl transcript as a
control. As seen in Lanes 5 and 6, when the bcr-abl RNA is exposed
to the ribozyme molecule of this invention, the RNA is broken into
two fragments of lower molecular weights, thereby rendering it
inactive.
DETAILED DESCRIPTION OF THE INVENTION
The compositions and methods of the present invention are designed
for the treatment of any leukemia or other cancer which is
characterized by the presence of a tumorigenic chimeric protein
such as that resulting from a chromosomal translocation, the gene
encoding the protein having a ribozyme cleavage site at or near the
chromosomal breakpoint. Exemplary disorders characterized by such a
protein include chronic myelogenous leukemia, acute lymphocytic
leukemia, as well as other leukemias, such as those involving c-myc
8q24 and bcl-1 and bcl-2.
The invention provides a synthetic RNA molecule or ribozyme. For
purposes of simplicity, the following description relates to a
novel RNA molecule of this invention designed for the cleavage of
the bcr-abl mRNA produced by the Philadelphia chromosome. However,
as noted above, the present invention is not limited to this
molecule, but encompasses other molecules designed according to
this invention for cleavage of similar genes in other cancers. The
novel ribozyme has been designed to cleave the selected target
chimeric gene after the ribonucleic acid sequence G-U-X, wherein X
is the ribonucleotides A, U or C.
To use ribozyme-based reactions as a potential therapy for human
leukemias, a hammerhead ribozyme motif that can cleave specifically
the bcr-abl RNA thereby blocking the synthesis of BCR-ABL protein,
the primary cause of these leukemias, is provided by this
invention. The design of the hammerhead is such that it can only
affect the oncogenic bcr-abl gene product without having any effect
on normal bcr and c-abl gene transcripts. By cleaving the bcr-abl
gene transcript, this ribozyme can prohibit synthesis of the
BCR-ABL protein associated with tumor cells and, return the cells
to normalcy.
The hammerhead ribozyme, which is preferred in this invention,
comprises separate sequences: a catalytic sequence and a substrate
binding sequence in two parts (the bcr-abl mRNA is the substrate).
The ribozyme is a synthetic, catalytic RNA of between 35-45
nucleotides in length. A presently preferred ribozyme is about 40
nucleotides in length. The designed ribozyme may vary in structure
so long as it contains the catalytic sequence. However, the
cleavage rates of the ribozyme will vary depending upon the
secondary (and possibly tertiary) structures of the hammerhead.
The hammerhead ribozyme of this invention comprises a single strand
of ribonucleic acids comprising:
(1) a sequence complementary to a sequence of the coding strand of
the hybrid gene 5' to the breakpoint of the translocation and
capable of hybridizing to that part of the gene (a first substrate
binding sequence);
(2) a second sequence complementary to a sequence of the coding
strand of the hybrid gene 3' to the breakpoint of the translocation
and capable of hybridizing thereto (a second substrate binding
sequence), and
(3) a sequence therebetween encoding a catalytic ribozyme domain
capable of cleaving the hybrid chromosome at or near the breakpoint
(the catalytic or ribozyme sequence).
Substrate binding sequences (1) and (2) above depend for their
structure on the sequence of the hybrid gene which is targeted for
destruction by the invention. These sequences are complementary to
appropriate parts of the hybrid gene which flank the translocation
breakpoint, e.g., the portion of the gene at which the two
chromosomes are fused. The function of these sequences of the RNA
molecule of this invention is to specifically isolate the hybrid
gene RNA and position the ribozyme (3) for cleavage of the
gene.
These sequences (1) and (2) are desirably between 5 to 11
nucleotides in length. More preferably, these flanking sequences
are between 6 and 10 nucleotides in length. The length of these
sequences provides a hybridization event with the target gene
sufficient to permit cleavage of the gene at or near the
breakpoint, thereby disabling the production of the hybrid
tumorigenic protein. After the gene is cleaved, the RNA molecule is
designed to dissociate from the pieces. The ability to release the
fragments of the cleaved gene relates also to the length of the
flanking sequences of the RNA molecule. The same RNA molecule may
then encounter another gene and perform the same cleavage function
repeatedly until the molecule is eventually degraded by the
cell.
In a specific embodiment, a ribozyme molecule of this invention has
a 40 nucleotide base sequence comprising ##STR1## wherein U is
Uridine, C is Cytosine, G is Guanidine, and A is Adenine.
Optionally, the ribozyme may be provided with a cap structure, e.g.
a 5'GpppG, which acts as a stabilizer in an intracellular
environment.
The sequence of the RNA oncogene bcr-abl, i.e., the site on the
bcr-abl gene transcript where the RNA molecule of this invention
will bind is: 5' A-G-C-A-G-A-G-U-U (cleavage
site)-C-A-A-A-A-G-C-C-C-U 3'. The ribozyme is designed so that
cleavage occurs after the 5' GUU 3' sequence of the bcr-abl mRNA.
FIG. 2 illustrates the binding of the ribozyme to the substrate
(the bcr-abl gene transcript).
The ribozyme of this invention can be prepared by chemical
synthesis or produced in recombinant vectors by conventional means
[see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual),
Cold Spring Harbor Laboratory (1982)].
As is described in exemplary form below, ribozyme RNA sequences may
be synthesized conventionally by means of, for example, the RNA
polymerase system such as T7 or SP6. bcr-abl mRNA substrates may be
cloned from, for example, K562 cell line (available from the
American Type Culture Collection, Rockville, Md., U.S.A., Accession
Number ATCC# CCL 243) or BV173, EM2, Nalm-1, EM3, and any CML cell
lines. The sequence of the bcr-abl gene is published in E.
Shtivelman et al, Nature, 315:550-553 (1985). The RNAs are
separated by electrophoresis and purified in acrylamide-urea
gels.
The mechanism by which the ribozyme works is as follows: the
substrate strand is designed in such a way that one half of it
hybridizes to the bcr sequence of the hybrid gene and the other
half of the substrate RNA binds to the abl sequence of the hybrid
gene. The catalytic sequence is thereby placed in proximity to the
5'GUU3'sequence of the site of the targeted hybrid gene. Upon
cleavage the bcr-abl gene transcript is destroyed, and the ability
of the gene to direct the synthesis of BCR-ABL protein is
interrupted. In the absence of the oncogenic protein, the cell
returns essentially to normal.
Because the ribozyme effects only site-specific cleavage, normal
cellular bcr and abl gene transcript sites therefore remain
unaffected by the action of the ribozyme. Thus, only cells
containing the translocation resulting in the Philadelphia
Chromosome are altered by this ribozyme.
Desirably for both production of the ribozyme of this invention and
to provide delivery systems for exposing bone marrow cells to the
ribozyme, recombinant vectors are employed. The ribozyme construct
may be placed in a viral vector, of which many are known to the
art. Viral vectors are preferred because they have the capacity to
infect the cell where the ribozyme must be located to function
appropriately. Presently preferred vectors are retroviral vectors,
adenoviral vectors, vaccinia vectors, and others, such as described
by E. Gilboa, Adv. Exp. Med. Biol., 241:29 (1988) and P. H. Pouwels
et al, "Vectors for Animal Cells", in Cloning Vectors: A Laboratory
Manual, ch.7 (Elsevier, Amsterdam: 1985). Other conventionally
employed vectors designed for use in mammalian, bacterial, yeast,
fungal or insect systems may be employed to recombinantly express
the ribozyme of this invention, but are not preferred for delivery
purposes into the cells.
The resulting viral vector is then exposed to the cells wherein the
cells become infected, the ribozyme is replicated and functions as
described above.
Alternative delivery systems for the ribozyme may employ other
known components, such as a lipid delivery system, first described
in P. L. Felgner et al, Proc. Natl. Acad. Sci. U.S.A., 84:7413-7417
(1987). The lipid functions to fuse with the cells allowing the
ribozyme to enter the cells. Transferrinfection of K562 cells,
recently shown to be an effective alternative to retroviral
infection, may be successfully employed as a delivery system
[Cotten et al, PNAS, 87:4033 (1990)]. Other delivery systems may
include direct addition of the ribozyme to cells, which has been
shown to be successful with antisense DNA oligomers [S. L. Luke et
al, PNAS, 86:3474-3478 (1989)].
The ribozyme of the present invention may thus be employed as a
therapeutic agent in the treatment of leukemias and other cancers
such as those characterized by the presence of the Philadelphia
Chromosome. The method according to this invention may also be used
in the treatment of other diseases, such as any leukemia or solid
tumor which results from and contains the fusion of at least two
normal genes, resulting in the generation of an abnormal chimeric
gene product, or otherwise characterized by the presence of other
chimeric gene products resulting from chromosomal translocations or
other fusion mechanisms, wherein the resulting gene has a 5'GUX3'
sequence close to the breakpoint. Other ribozymes may be designed
according to this method for use in analogous treatments for these
other cancers.
Preferably this treatment is accomplished by contacting bone marrow
cells extracted from a patient ex vivo with a sufficient number of
ribozymes, or vectors carrying ribozymes, for a time sufficient to
effect cleavage of the oncogene present in the cells.
Alternatively, the method may employ contacting the cells in vivo,
for example, by administration directly into the bone marrow of a
leukemic patient of vectors carrying the RNA molecule of the
invention. When either method is applied to the cells of CML
patients, the RNA molecule of this invention causes the specific
destruction of the bcr-abl mRNA, resulting in the loss of synthesis
of the tumorigenic BCR-ABL protein.
Once transmitted into the cellular environment via a vector, the
ribozyme is expected to survive within the cell for a short period
of time. Since there is not necessarily only a one to one
relationship between the ribozyme and the target oncogene, a single
ribozyme is expected to bind and cleave a number of oncogenic
transcripts before being degraded or destroyed by the natural
enzymes in the cells and/or the natural functions of the immune
system.
The patient may be treated with conventional chemotherapy or
radiation to substantially destroy the remaining bone marrow cells
carrying the bcr-abl oncogene, and the treated cells are then
returned to the patient. The treated cells, when returned to the
patient may then be stimulated by various known hematopoietic
growth factors to repopulate the bone marrow with cells which do
not carry the oncogenic transcript.
This method has the advantage over conventional cancer or leukemia
treatments, such as bone marrow transplant, of avoiding
complications caused by lack of compatibility and rejection because
in the present method only the patient's own cells are involved.
Further, it is expected that many of the side effects associated
with conventional chemotherapy may be avoided because the treatment
takes place essentially ex vivo. However, this method may also be
used in connection with other treatments, such as radiation or
chemotherapy.
The in vitro reaction depicted in FIG. 3 indicates the
applicability of the reaction to the in vivo conditions. The
reaction was performed at 37.degree. C. under physiological pH of
7.5 with the presence of the Mg++ cofactor. Under these conditions
enzymatic activity will occur until the elimination of either the
substrate or degradation of the ribozyme.
The number of ribozymes which are required can be determined in
vitro by titration of a known substrate with known riboxyme until
no activity is observed.
The following examples illustrate the compositions and methods of
this invention.
Example 1 - Ribozyme RNA Synthesis
The ribozyme was synthesized as an oligonucleotide on an Applied
Biosystems DNA synthesizer. The 40 mer ribozyme was purified by
isolation from 20% polyacrylamide-7 M urea gels. The products are
located by either autoradiography or UV shadowing. The resultant
products band is crushed, and soaked for between 1 hour and
overnight in 2 volumes of 0.5M NaOAc pH 7.0. The extracted RNA is
concentrated by ethanol precipitation and resuspended in H.sub.2 O.
Purified RNAs are stored in H.sub.2 O at -20.degree. C.
The resulting ribozyme was 40 nucleotides in length with the
following sequence: ##STR2## wherein U is Uridine, C is Cytosine, G
is Guanidine, and A is Adenine.
Example 2 - Preparing the bcr-abl Substrate
The 5' end of bcr-abl RNA containing the bcr-abl breakpoint was
expressed by inserting a 420 bp fragment of bcr-abl cDNA, which was
constructed from a library constructed from K562 mRNA, into the
plasmid vector pGEM5 (Promega). This sequence is transcribed in
vitro using T7 RNA polymerase in the presence of .sup.32 P-rCTP.
The resulting labelled RNA was isolated and purified.
The vector containing the BCR-ABL fragment was linearized by NdeI
digestion and transcribed in vitro using T7 RNA polymerase in the
presence of .sup.= P-rCTP. The reaction was stopped after one hour
at 37.degree. C. by the addition of DNASE. Following digestion of
the plasmid template, labelled RNA transcripts were loaded onto a
4% PAGE-7M urea gel and isolated by elution of the band from the
gel slice. The purified RNA transcript was resuspended in H.sub.2 O
and stored at -20.degree. C. until use.
The T7 polymerase generated RNA transcript was 499 bases in length;
with 73 bases of pGEM 5 polylinker sequence prior to the 420 bases
of BCR/ABL substrate and 6 bases from the polylinker restriction
linearization site. The GUU cleavage site is located 140 bases from
the 5' end of the BCR/ABL specific sequences.
Analysis of the RNA transcript was used to characterize the
cleavage products which would be expected if the assay described
below in Example 3 proved the efficacy of the ribozyme. The 5'
cleavage product should be 140+73=213 nucleotides and the 3'
cleavage product should be 280+6=286 nucleotides. The intact
transcript is 73+420+6=499 nucleotides. In the absence of cleavage,
one specie is expected: an intact 499 nucleotide fragment extending
from the T7 polymerase start site, to the end of the linearized
plasmid. This includes the BCR/ABL segment as well as sequences
from the pGEM5 expression plasmid. If cleavage of the bcr-abl RNA
occurs, the 499 fragment is split into two fragments 213 and 286
nucleotides in length.
From this it was determined that the uncleaved bcr-abl gene
transcript was 499 nucleotides in length. This RNA was used as a
substrate in the test of Example 3 below.
Example 3 - Specificity of the Cleavage Site
To assess whether the synthetic ribozyme of Example 1 can cleave
the bcr-abl mRNA substrate, the ribozyme of Example 1 and the RNA
substrate of Example 2 (1 pmol of each) were mixed in a 10-.mu.l
reaction volume containing 50 mM Tris-HCl, pH 7.5. This resembles
the physiologic pH of 7.4. As a control the MgCl.sub.2 is omitted
and 10 mM EDTA is added as the typical ribozyme-mediated cleavage
is dependent on metal ions such as Mg++. The mixture is heated to
95.degree. C. for 2 minutes and quick-cooled on ice. 10 mM
MgCl.sub.2 is added and then the mixture is incubated at 37.degree.
C. for 14 hours.
Following the incubation, the reaction products were analyzed on a
4% polyacrylamide gel under denaturing conditions. The results
presented in FIG. 3 show that the synthetic hammerhead cleaves the
substrate RNA containing the bcr-abl junction, in the expected
manner.
After cleavage, the bcr-abl fragment sizes were 290 nucleotides and
160 nucleotides. [See lanes 5 and 6 in the gel of FIG. 3]. These
results also show that the cleavage occurs very efficiently in the
presence of Mg++ ions. In contrast, this cleavage reaction was
inhibited by EDTA. These results indicate that the introduction of
this synthetic RNA ribozyme into leukemic cells synthesizing
bcr-abl gene transcripts will result in the specific degradation of
bcr-abl RNA which, in turn, prevents the synthesis of the oncogene
BCR-ABL. Thus, this treatment is expected to result in the reversal
of the leukemic process.
Example 4 - Production and Use of a Vector for Delivery of the
Ribozyme to Cells
To deliver the ribozyme to a patient's leukemic cells, the ribozyme
is inserted as a DNA fragment into a retroviral expression vector.
A synthesized DNA fragment, such that its expression will result in
the active ribozyme molecule, is cloned into a retroviral vector by
standard methods (T. Maniatis et al, cited above). The retroviral
vector, pC-1, contains the Moloney murine leukemia virus LTRs and a
selectable neo gene. The crippled vector allows packaging and
infection of the inserted gene. However, because the pathogenic
portions of the parent virus have been removed, the pathogenicity
of the parental viral infection has been eliminated. The
recombinant virus containing the ribozyme DNA sequence is
transfected into a packaging cell-line, such as PA31I7 or .psi.2,
to generate a producer cell line.
Bone marrow cells from a leukemic patient are infected either by
co-cultivation with the producer cell-line or by incubation with
cell-free virus containing supernatant. Following overnite
infection, the bone marrow cells are washed in PBS and grow in
vitro for a period of 14-21 days. Successfully infected cells are
selected for by the inclusion of G418 (Gibco) in the medium at two
days post-infection. The presence of the neo gene in the
ribozyme-containing retrovirus allows for growth in the presence of
this compound. Therefore cells which were not infected die. The
population of infected bone marrow cells are transplanted into the
patient. Bone marrow cells and/or peripheral blood samples are
monitored by the PCR-RT technique using BCR-ABL specific primers to
show the presence or absence of BCR/ABL transcripts and the success
of the treatment.
Numerous modifications and variations of the methods of this
invention are expected to occur to those of skill in the art. For
example, the methods described above may be employed to design
other ribozymes suitable for the treatment of other leukemias.
Similarly, other recombinant techniques, vectors and methods for
assembling the ribozymes may be selected by one of skill in the art
without departing from this invention. Thus, the invention and such
modifications are encompassed by the appended claims.
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